Clearing agents for histological tissue



Patented July 14, 1953 j 2,645,618 CLEARING AGENTS FOR HIsToLoGIcAL TISSUE Andrs Ferrari, Jr., Uniond ale, N. Y., assignor to Tcchnicon Chemical Company, Inc., New York, -N. Y., a corporation-of New York N Drawing. Application fiecember 6, 1950,

' Serial No. 199,501

This invention relates to the preparation of histological tissue specimens for microscope examination, and more particularly to clearing agents employed in treating the tissue in the course of such preparation.

The preparation of tissue to enable the'microscopic'examination thereof involves a series of treatments of the tissue prior to the cutting of the sections from the tissue specimens for the staining and mounting of the sections on the microscope slides: More particularly, in the preparation of the tissue itis necessary to immerse the tissue successively in aseries of liquid agents for certain lengths of time, first to fix the tissue, then to wash the same for removing the fixative, then to dehydrate the tissue, usually by immersion of the tissue successively in alcohols or other dehy- As pathologists are well aware, the purpose of treating tissue with a clearing agent before infiltrating the tissue with parafiin is ;to eliminate from the tissue all liquid which, ifnot removed, would decrease the refractive index of the tissue and would interfere with the thorough infiltration of the paraflininto the tissue resulting in poorly cut tissue sections. Among pathologists it is recognized that the higher the refractive index, i. e., the transparency, of the tissue the more easily and accurately it can be examined microscopically. It is of the utmost importance, therefore, that a tissue-clearing agent be capable of removing all such liquid from the tissue and have a high degree of solvency for, or miscibility with, parafiin so as to facilitate theinfiltrationof the paraffin into the tissue. It is also very important that the clearing agent be of such character that it does not harden the tissue, does not impair the cytological structures of the-tissue, is volatile at temperatures of 120 to 135 F., and is substan-,

tially non-inflammable under the conditions of use. Substantial volatility at temperatures about 120 to 135 F. is important from the standpoint of the efiicient operation of the automatic immersion apparatus. The use of a clearing agent which is non-volatile at temperatures about 120 to 135 5; Claims. (01. 252 ss4) V 2 F. results in the accumulation of clearing agent in the paraffin bath in which the tissue is immersed after treatment with the clearing agent solution;

Such accumulation detrimentally affects the homogeneity of the paraffin' bath which in turn re sults in unsatisfactory infiltration of the tissue with the paraffin. The use of a clearing agent which is substantially non-inflammable under the conditions of use is important from the standpoint of eliminating a fire hazard.

The primary object of the present invention is to provide a clearing agent which has the follow-.

ing properties:

1. It has a high solvent power for liquid present in the tissue.

2. It does not harden the tissue and does not.

impair the cytological structure thereof.

3. It ha a high solvent power for ormiscibility with parafiin and thus facilitates the infiltration of the paraffin into the tissue.

4. It does not decrease the refractive index the tissue.

5. It is substantially non-inflammable under the conditionsof use.

6. It has substantial volatility at temperatures about to F.

Other objects and advantages of this invention carbon having from 2 to 4 carbon atoms, liquidat atmospheric temperature, and carbon tetrachloride. These constituents are mixed in any desired order at room temperature in the propor-' tions of 10% to 40%, preferably 20% to 30%, liquid chlorinated naphthalene, 20%-80%, prefer ably 60%-40 liquid chlorinated aliphatic hydrocarbon and 10% to 40%, preferably 20% to 30% carbon tetrachloride. All proportions in this specification are by volume.

The chlorinated naphthalene used is an oilj liquid at atmospheric temperature, having a white to pale straw color, a specific gravity at 770 F. of 1.19 to 1.25, a viscosity (Saybolt Universal seconds at 77 F.) of 33 to 37, a distillation range (ASTM F.) 480 to 590, a flash point' (ASTM Cleveland open cup F.) 203, and a fire point (ASTM Cleveland open cup F.) 338. The

preferred chlorinated naphthalene is'that sold under the trade namefI-Ialowax 1000 by the Halowax Products Division of'Union Carbide and;

Carbon Corporation. 7

The chlorinated aliphatic hydrocarbon used is ethylene dichloride, ethylene trichloride, trichlorpropane or other chlorinated aliphatic hydrocarbon having good solvent properties for fats and waxes and compatible with liquid chlorinated naphthalene. Ethylene dichloride is preferred because of its ready availability, its high solvent power for paraffin and liquids present in the tissue and its excellent compatibility with liquid chlorinated naphthalene. The use of ethylene dichloride in the proportions indicated results in an efficient clearing agent of reduced cost.

The carbon tetrachloride raises the flash point of the mixture and this without detrimentally affecting the solvent properties of the liquidchlo rinated naphthalene and chlorinated aliphatic hydrocarbon for parafiin or for the liquids present in the tissue.

The liquid chlorinated aliphatic hydrocarbon when mixed .with the liquid chlorinated naph- Percent Ethylene dichloride 60 Carbon tetrachloride Liquid chlorinated naphthalene (Halowax This clearin agent when subjected to a flash point test (ASTM Cleveland open cup) gave ofi sumcient chlorine at 80 C. to extinguish the flame. It has a distillation range as follows:

Initial boiling point 77 C. 50% ofi 815 C.

Dry point 297.0 C.

Its refractive index at C. is 1.4886 and its specific gravity 20/20 C. is 1.319.

It will be understood, that in using the clearing agent of the present invention, the tissue is immersed therein after the dehydration treatment and before the paraffin infil tration process. The clearing agent of this invention involving the three constituents above enumerated has been found to combine to a' surprising and exceptional extent the six properties hereinabove enumerated and has been found to be an exceptionally efficient clearing agent. has a pleasant odor. Further, it is a materially better solvent for paraflin than butyl acetate which has heretofore been used as a clearing agent. In these respects it is far superior to butyl acetate.

Tissue blocks comprising tissue which has been treated with the clearing agent of this invention have greatly improved cutting qualities, the cytological structures of the tissues are effectively preserved, and the appearance of the tissue sections is considerably improved.

It will be understood that various change may be made in the clearing agent of the present invention within the skill of the art, without. de-

parting from the underlying idea or principles of' my invention within the scope of theappendedclaims. Y

Having thus described my invention, what I claim and desire to secure by Letters Patent, is:

1. A clearing agent for use in the preparation of histological tissue for microscopic examination, consisting essentially of from 10% to by volume of a chlorinated naphthalene liquid at atmospheric temperature, having a specific gravity at 77 F. of 1.19 to 1.25, a viscosity (Saybolt Universal seconds at 77 F.) of 33 to 37 and a distillation range (ASTM) of 480 F. to 590 F., from 10% to 40% by volume of carbon tetrachloride and from 80% to 20% by volume of a chlorinated aliphatic hydrocarbon having from 2 to 4 carbon atoms and liquid at atmospheric temperature.

3'. A clearin agent for use in the preparation of histological tissue for microscopic examination, consisting essentially of from 20 to by volume of a chlorinated naphthalene liquid at atmospheric temperature, having a specific gravity at 77 F. of 1.19 to 1.25, a viscosity (Saybolt Universal seconds at 77 F.) of 33 to 37 and a distillation range (ASTM) of 480 F. to 590 F., from 60% to of a chlorinated aliphatic hydrocarbon from the group consisting of ethylene dichloride, ethylene trichloride and trichlorpropane, and from 20% to 30% of carbon tetrachloride.

4. A clearing agent for use in the preparationof histological tissue for microscopic examination, consisting essentially of from 20% to 30%.

by volume of a chlorinated naphthalene liquid at atmospheric temperature, having a specific gravity at 77 F. of 1.19 to 1.25, a viscosity (Saybolt Universal seconds at 77 F.) of 33 to 37 anda distillation range (ASTM) of 480 F. and 590? F., from to 40% ethylene dichloride and from 20% to 30% carbon tetrachloride.

5. A clearing agent for use in the preparation of histological tissue for microscopic examination consisting essentially of about 60% ethylene dichloride, about .20% carbon tetrachloride and about 20% of a chlorinated. naphthalene liquid at atmospheric temperature, having a specific gravity at 77 F. of 1.19 to 1.25., a viscosity (Saybolt Universal seconds at 77 F.) of 33 to 37 and a distillation range (ASTM) of 480 F. to 590 F.

. ANDRES FERRARI, JR.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,850,881 Johnsen Mar. 22, 1932 2,017,327 Sullivan Oct. 15, 1935 2,393,580 Weiskopf Jan. 22, 1946 OTHER REFERENCES Wadsworth, Standard Methods, 3d edit. pp. 

5. A CLEARING AGENT FOR USE IN THE PREPARATION OF HISTOLOGICAL TISSUE FOR MICROSCOPIC EXAMINATION CONSISTING ESSENTIALLY OF ABOUT 60% ETHYLENE DICHLORIDE, ABOUT 20% CARBON TETRACHLORIDE AND ABOUT 20% OF A CHLORINATED NAPHTHALENE LIQUID AT ATMOSPHERIC TEMPERATURE, HAVING A SPECIFIC GRAVITY AT 77* F. OF 1.19 TO 1.25 A VISCOSITY (SAYBOLT UNIVERSITY SECONDS AT 77* F.) OF 33 TO 37 AND A DISTILLATION RANGE (ASTM) OF 480* F. TO 590* F. 